1. Field of the Invention
The present invention relates to a biological test method using cells, which is used in the fields involving pharmaceuticals, medical care, foods, medicine, pharmacy, biology, and the like. The present invention particularly relates to a biological test method using three-dimensional culture considered to enable obtainment of information that is more useful than that obtained via conventional two-dimensional culture. The biological test method is technology that is worthwhile as an alternative to animal testing.
2. Background Art
In recent years, lower investment efficiency in research and development has become an issue in the pharmaceutical industry. Hence, biological test methods using three-dimensional culture methods are attracting attention as methods useful for reducing the high costs of animal experimentation. Such biological test methods using three-dimensional culture methods are expected to be useful for obtainment of basic data that enable in vivo prediction of the absorption, distribution, metabolism, excretion, and toxicity of new drugs in particular. This technology will influence not only the pharmaceutical industry, but also on treatment and diagnosis in the fields of medical care, food safety tests, and the like. Also, rapidly developed from research using a conventional two-dimensional culture method is research using a combination of cellular imaging technology and a three-dimensional culture method. Practical use of screening technology that constitutes a combination of cellular imaging technology and a three-dimensional culture method has already been initiated.
A conventional three-dimensional culture method will be described below. The most simple three-dimensional culture method involves culturing cells dispersed in a gel matrix such as collagen or agarose gel in an appropriate container. Furthermore, a method that involves forming a gel layer on the bottom of a culture container, seeding cells on the bottom, and then culturing the cells is also regarded as a three-dimensional culture method when the cells migrate or infiltrate into the gel. Non-patent Document 1 discloses a method, namely a sandwich culture method that involves forming a gel layer on the bottom of a culture container, seeding cells on the gel layer, culturing the cells, removing medium after a predetermined time period, adding a gel precursor onto the cell layer for gelling, and then further culturing the resultant. Furthermore, Non-patent Document 2 discloses a method that involves forming a gel layer on a general two-dimensional culture cells and then performing a test. Non-patent Document 3 discloses a method that involves previously culturing cells on the surfaces of beads, embedding them within a gel matrix, and then performing a test. Moreover, Non-patent Document 4 discloses a method that involves embedding cell spheroids within a gel matrix and then performing a test. These three-dimensional culture methods are attracting attention concerning the culture of a plurality of cell types; that is, coculture. That is because cell behavior more analogous to that observed in vivo can be expected with the use of coculture compared with culture of a single cell type.
However, these methods are problematic in that the initial position coordinates of single cells or cell aggregates cannot be specified and control of the distribution of cell aggregate sizes is difficult. Hence, application of these methods to an advanced biological test system using a combination of robotics and viable cell analysis techniques has been limited. The use of the method disclosed in Non-patent Document 4 basically addresses the above problems since one spheroid is formed in each well of a formatted well plate. However in this case, a plurality of spheroids cannot be cultured with determined position coordinates thereof in each well, so that obtainment of a plurality of data from each well and statistical data analysis cannot be performed. Furthermore, a method must be employed to observe spheroids being embedded in gel under a microscope, which involves harvesting one spheroid prepared per well of a well plate and then dispersing it for casting in a solution before gelling. Furthermore, the form of a cell aggregate that can be used in the method disclosed in Non-patent Document 4 is limited to a spheroid.
Meanwhile, in recent years, the field of research using a combination of a fine processing technology such as semiconductor technology or printing technology and biotechnology has been significantly developed. There are great expectations for this research, since it may lead to development of new healthcare technology or production of efficient drug discovery tools. For example, as disclosed in Non-patent Document 5, Patent Document 1, Patent Document 2, and Patent Document 3, it has been reported that a culture instrument is produced via application of technology for lithographying a photosensitive polymeric material (photoresist) or the like, so as to obtain a two-dimensional cell pattern. Furthermore, Patent Document 4 and Patent Document 5 disclose technology that realizes a two-dimensional co-culture system through combination with a step of removing a photoresist, so as to use the system for a cell function test. Recently, technology for preparing a two-dimensional cell pattern through application of coating with a thickness at the molecular level and the application of such technology have been reported. Non-patent Document 6 discloses technology for testing cell movement with the use of a cell pattern having a cell having an artificially controlled adhesion mode as a component. Moreover, Non-patent Document 7 discloses technology for testing cell proliferation activity or the like using an artificial cell pattern containing cell aggregates as components or using a cell sheet that is formed on an instrument having artificial geometric up and down. Furthermore, Non-patent Document 8 reports a test method that is not an example of using a cell pattern and involves causing a microgel array on which a metabolism enzyme is complexed with a chemical substance to come into contact with monolayered culture cells, following which the toxicity of a metabolite produced by the metabolism enzyme is tested.
Among these examples of technology, a general method using a polymer resist (Non-patent Document 5, Patent Document 1, Patent Document 2, or Patent Document 3) often leads to the production of cell movement test systems differing from general two-dimensional culture, since the resist generally has a degree of thickness that hinders cell movement. The resulting test systems are often considered to be troublesome artificial test systems and they have failed to attract attention in application fields such as pharmacological tests. In the meantime, the above problem concerning such resist thickness has been addressed in Patent Document 4, Patent Document 5, Non-patent Document 6, and Non-patent Document 7. However, these documents do not mention any biological test method that involves culturing an artificial single cell pattern or a cell aggregate pattern in a matrix. Technology disclosed in Non-patent Document 8 is expected as a new high-throughput pharmacological test method. However, this is not appropriate as a test method for testing parameters concerning single cell or cell aggregate movement.
Furthermore, technology that is a combination of fine processing technology and three-dimensional culture has been recently reported. Patent Document 6 discloses a method for preparing a spheroid pattern comprising artificially prepared vascular endothelial cells and hepatic cells and the use of the method. Non-patent Document 9 discloses technology that involves artificially and three-dimensionally aligning cells in a matrix before gelling and then gelling the matrix. Moreover, Patent Document 7 discloses a method for preparing artificial tissue, which involves transferring a cell layer prepared via pattern culture to a complex composed of a cell layer and a basal membrane layer, so as to prepare artificial tissue. Non-patent Document 10 discloses a method that involves pattern culturing cells on collagen gel using a stencil mask, removing the stencil mask, placing a collagen solution on the cell pattern for gelling, and then sandwich culturing the patterned cells. Furthermore, Non-patent Document 11 discloses technology that involves preparing a polymer pattern through laser ablation, pattern culturing vascular endothelial cells, transferring the cell pattern onto gel, forming gel on the transferred cell pattern, and performing three-dimensional culture.
However, Patent Document 6 does not disclose any biological test method that involves three dimensional culturing of an artificially prepared single cell pattern or cell aggregate pattern in a gelled matrix. Non-patent Document 9 discloses technology for culturing a cell pattern artificially prepared in gel, but does not disclose any method for testing a biological indicator concerning the movement of single cells or cell aggregates prepared in an artificial pattern in a gel matrix. Patent Document 7, Non-patent Document 10, and Non-patent Document 11 disclose three-dimensional culture technology for cell aggregates prepared in an artificial pattern. However, these inventions are not intended for performance of biological tests for parameters concerning the movement or the proliferation of cell aggregates, and they disclose almost nothing concerning such parameters. Furthermore, according to Patent Document 7 and Non-patent Document 11, transfer of cells from a hard culture instrument requires approximately 24 hours. This suggests that cells strongly interact with such a hard culture instrument. Hence, technology according to Patent Document 7 and Non-patent Document 11 is problematic in that it is likely to be recognized as a culture system that includes artificial factors, compared with conventional three-dimensional culture technology. Moreover, the method according to Non-patent Document 10 is problematic in that freedom to design a cell pattern is limited since use of a stencil mask is an essential requirement.    Patent Document 1 JP Patent Publication (Kokai) No. 3-7576 A (1991)    Patent Document 2 JP Patent Publication (Kokai) No. 5-176753 A (1993)    Patent Document 3 JP Patent No. 2777392    Patent Document 4 U.S. Pat. No. 6,133,030    Patent Document 5 U.S. Pat. No. 6,221,663    Patent Document 6 WO2003/010302    Patent Document 7 JP Patent Publication (Kokai) No. 2005-342112 A    Non-patent Document 1 The FASEB Journal, vol. 10, 1471-1484 (1996)    Non-patent Document 2 Molecular Biology of the Cell, vol. 13, 2474-2485 (2002)    Non-patent Document 3 Tissue Engineering, vol. 11, no. 1/2, 257-266 (2005)    Non-patent Document 4 The FASEB Journal, vol. 15, 447-457 (2001)    Non-patent Document 5 Journal of Biomedical Materials Research, vol. 32, 165-173 (1996)    Non-patent Document 6 Proceedings of the National Academy of Sciences, vol. 102, no. 4, 975-978 (2005)    Non-patent Document 7 Proceedings of the National Academy of Sciences, vol. 102, no. 33, 11594-11599 (2005)    Non-patent Document 8 Proceedings of the National Academy of Sciences, vol. 102, no. 4, 983-987 (2005)    Non-patent Document 9 Nature Methods, vol. 3, no. 5, 369-375 (2006)    Non-patent Document 10 Journal of Biomedical Material Research, vol. 52, 346-353 (2000)    Non-patent Document 11 Report of Research Support 2000 (ISSN 0916-3719) Japan Cardiovascular Research Foundation